Electroporation and electrophoresis system and method for achieving molecular penetration into cells in vivo

ABSTRACT

The electroporation system and method combine pulses having different characteristics for delivering molecules to cells in vivo. The pulses include a high-intensity pulse for inducing electroporation and a low-intensity pulse to induce electrophoretic molecule movement within an interstitial space, molecule adherence to a cell membrane, and electrophoretic movement of the molecule through the permeabilized membrane. The use of a high-intensity and a low-intensity pulse achieves improved delivery; reduction of intensity and/or duration of pulses for inducing electroporation; and decreased muscle stimulation, tissue damage, and patient discomfort.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of patent application Ser.No. 10/708,111 of the same title by the same inventor, filed Feb. 10,2004 and now abandoned, which is a divisional application of patentapplication Ser. No. 09/507,859 filed Feb. 22, 2000 now issued U.S. Pat.No. 6,714,816.

FIELD OF INVENTION

The present invention relates to methods and apparatus for deliveringmolecules into a target cell, and, more particularly, to such methodsand apparatus for achieving such delivery into cells in vivo throughelectroporation and electrophoresis.

BACKGROUND OF THE INVENTION

The effect of electromagnetic fields on cell membranes has been studiedsince the 1960s. Early research focused on describing observations thatan applied electric field can reversibly break down cell membranes invitro. Throughout the 1970s the topic was more common in the literatureand continued to focus on describing the phenomenon that resulted frombrief exposure to intense electric fields as well as the entry ofexogenous molecules to the cell interior as a result of membranebreakdown. Applications began to emerge along with a betterunderstanding of reversible membrane breakdown in the 1980s.

Prior research led to the current understanding that exposure of cellsto intense electric fields for brief periods of time temporarilydestabilized membranes. This effect has been described as a dielectricbreakdown due to an induced transmembrane potential, and was termed“electroporation,” or “electropermeabilization,” because it was observedthat molecules that do not normally pass through the membrane gainintracellular access after the cells were treated with electric fields.The porated state was noted to be temporary. Typically, cells remain ina destabilized state on the order of minutes after electrical treatmentceases.

The physical nature of electroporation makes it universally applicable.A variety of procedures utilize this type of treatment, which givestemporary access to the cytosol. These include production on monoclonalantibodies, cell-cell fusion, cell-tissue fusion, insertion of membraneproteins, and genetic transformation. In addition, dyes and fluorescentmolecules have been used to investigate the phenomenon ofelectroporation. A notable example of loading molecules into cells invivo is electrochemotherapy. The procedure utilizes a drug combined withelectric pulses as a means for loading tumor cells with an anticancerdrug, and has been performed in a number of animal models and inclinical trials by the present inventors. Also, plasmid DNA has beenloaded into rat liver cells in vivo (Heller et al., FEBS Lett. 389,225-28). The methods published thus far for all in vivo applicationsutilize multiple direct current pulses that are substantially identical(single amplitude, single pulse duration, and a single duty cycle fordelivery). It is known that temporary permeabilization of cell membranescan result if the effects of pulsation fall within two thresholds.First, the intensity of the applied pulses must be above a thresholdvalue in order to electropermeabilize the membranes. This value iscell-type dependent. Second, if the intensity of the treatment is toohigh, then cells will be killed as a result of membrane damage, whichcan negate any desired effect of a molecule introduced into the cell.Thus it is critical to apply pulses with intensities that are above thedescribed lower threshold but below the upper threshold in order toimpart a temporary permeabilized state.

Protocols for the use of electroporation to load cells in vitrotypically use a suspension of single cells or cells that are attached ina planar manner to a growth surface. In vivo electroporation is morecomplex because tissues are involved. Tissues are composed of individualcells that collectively make up a three-dimensional structure. In eithercase, the effects on the cell are the same.

FIGS. 1A-1E illustrate details of the electrical treatment procedure.The method comprises:

1. A living biological cell in an electrically conductive medium ispositioned between two electrodes (FIG. 1 A). The cell's restingtransmembrane potential is indicated by + and − signs to indicate theseparated ionic species that make up the potential.

2. Application of an electrical field (in the form of an appliedpotential +V) between the two electrodes causes accumulation of chargeon either side of the cell. The separated charge adds to the restingpotential, resulting in an overall transmembrane potential (resting plusinduced). This charge will accumulate as the applied field is increasedup to a critical threshold value that is cell-type dependent.

3. If the overall transmembrane potential is increased above thisthreshold, by applying a field with sufficient magnitude indicated bythe pulse +V (FIG. 1C), then the cell membrane is dielectrically brokendown. This membrane breakdown has been termed electroporation and/orelectropermeabilization. Cells electroporate preferentially in themembrane region that faces the +electrode as the accumulation of chargein this membrane region adds directly to the resting potential. Lessporation takes place in the opposite side of the cell because theaccumulation of induced charge first cancels the resting potential andthen accumulates locally to form an induced potential. Thus a lowertotal transmembrane potential is induced on this side of the cell, whichresults in a lower degree of poration.

4. Immediately after electroporation, there is a rapid depolarization ofthe membrane that takes place as a result of the aqueous electropores(FIG. 1 D).

5. Normal membrane fluidity allows the electropores to seal in a timeframe that is approximately on the order of minutes (FIG. 1E).

The loading of molecules by electroporation in vitro as well as in vivois typically carried out by first exposing the cells or tissue ofinterest to a drug or other molecule (FIG. 2A). The cells or tissue arethen exposed to electric fields by administering one or more directcurrent pulses (FIG. 2B). Electrical treatment is conducted in a mannerthat results in a temporary membrane destabilization with minimalcytotoxicity. The intensity of electrical treatment is typicallydescribed by the magnitude of the applied electric field. This field isdefined as the voltage applied to the electrodes divided by the distancebetween the electrodes. Electric field strengths ranging from 100 to5000 V/cm have been successfully used, as reported in the literature,for delivering molecules in vivo and are also specific to the cells ortissue under investigation. Following cessation of stimulation, thepores reseal, with the desired molecules inside the cell (FIG. 2C).

Pulses are usually rectangular in shape; however, exponentially decayingpulses have also been used. The duration of each pulse is called pulsewidth. Molecule loading has been performed with pulse widths rangingfrom microseconds (μs) to milliseconds (ms). The number of pulsesdelivered has ranged from one to eight. Typically, multiple identicalpulses of the order of microseconds in duration are utilized duringelectrical treatment.

For molecules to be delivered to the cell interior by electroporation,it is critical that the molecule of interest be near the exterior of thecell membrane at the time of electroporation. It is also critical tohave molecules near all cells within a treated tissue volume in order toprovide efficient delivery to all cells within the treatment volume.Currently, molecules are injected intravenously, intraarterially,directly into the treatment site to provide a supply of molecules in theextracellular spaces of the tissues for delivery into the intracellularspaces by electroporation. Other methods for introducing the moleculesinto the extracellular spaces of tissues such as jet injection andparticle bombardment can be used to provide a source of molecules in theextracellular space for delivery using electrical fields.

Currently known delivery methods utilize the electrode systems outlinedpreviously using one or more pulses that are substantially identicalwith respect to their intensity (V/cm), pulse width, and duty cycle (ifmore than one pulse). Although protocols that used these types ofelectrodes and pulses to deliver molecules in vivo have been successful,there are some drawbacks. Pulsing protocols for delivering smallmolecules such as drugs, in particular, chemotherapeutic agents, haveused fields in the range of 1000-5000 V/cm. Such a high-intensity fieldcan cause patient discomfort in the form of pain and/or involuntarymuscle movement. Such drug-delivery protocols typically use multiplepulses on the order of microseconds in duration.

There are two types of pulsing protocols that have been used fordelivering DNA. The first type is identical to those pulsing protocolsused to deliver drugs and suffers from the same patient- relateddrawbacks. The second type uses pulses on the order of 100-800 V/cm,with pulses lasting up to hundreds of milliseconds. The drawback ofthese pulses is that their duration can cause great discomfort and alsotissue damage.

SUMMARY OF INVENTION

It is therefore an object of the present invention to provide animproved system and method for delivering molecules to cells in vivo.

It is an additional object to provide such a system and method that donot produce the discomfort and tissue damage associated with previouslyused devices and methods.

These objects and others are achieved by the system and method of thepresent invention, which combine pulses having different characteristicsdelivering molecules to cells in vivo. The pulses of the presentinvention comprise a high-intensity pulse for inducing electroporationand a low-intensity pulse to induce electrophoretic molecule movementwithin an interstitial space, molecule adherence to a cell membrane, andelectrophoretic movement of the molecule through the permeabilizedmembrane.

The molecules, which may comprise a unitary charged molecule or aplurality of different charged molecules, may be delivered by a methodknown in the art, such as by injection combined with electroporation,particle bombardment, and jet injection, although these methods are notintended as limitations. The types of molecules may include, but are notintended to be limited to, amino acids, bioactive molecules,polypeptides, proteins, antibodies (or fractions thereof),glycoproteins, enzymes, nucleic acids, oligonucleotides, RNA, DNA,competent DNA, plasmid DNA, chromosomes, drugs, other charged organic orinorganic molecules that may or may not have a localized charge region.

The use of a high-intensity and a low-intensity pulse achieves:

1. Equal or better in vivo delivery over conventional pulsing protocolsusing a single type of pulse.

2. Reduction of intensity and/or duration of pulses for inducingelectroporation over conventional pulsing protocols using onlyhigh-intensity pulses.

3. Decreased muscle stimulation, tissue damage, and patient discomfort.Target tissue may include, but is not intended to be limited to, normalor abnormal cells within tissues, skin, tumor tissue, or muscle.

The features that characterize the invention, both as to organizationand method of operation, together with further objects and advantagesthereof, will be better understood from the following description usedin conjunction with the accompanying drawing. It is to be expresslyunderstood that the drawing is for the purpose of illustration anddescription and is not intended as a definition of the limits of theinvention. These and other objects attained, and advantages offered, bythe present invention will become more fully apparent as the descriptionthat now follows is read in conjunction with the accompanying drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1A-1E (prior art) Conceptual two-dimensional depiction ofelectroporation of a cell subjected to an electromagnetic field. Regionsof membrane breakdown, depicted as pores, are formed at the ends ofcells facing the electrodes. Electromagnetic field exposure is achievedby applying a potential between electrodes − and +.

FIG. 2 (prior art) The process of delivering molecules byelectroporation. FIG. 2A. Cells in vitro or in vivo are exposed to themolecule of interest. FIG. 2B. Direct current pulses are administered tothe cells to cause a temporary membrane destabilization that allows themolecules to more freely enter the cell interior. FIG. 2C. Cells returnto their normal state after pulsation, leaving the molecule within thecells.

FIG. 3 is a schematic diagram of an exemplary pulsing sequence forachieving the delivery of molecules into cells in vivo.

FIG. 4 shows mean quantitative expression data for luciferase in normalmurine skin following application of a control and three differentsequences of pulsed fields.

FIGS. 5A-5E illustrate a possible sequence of events leading to thesynergistic effect of combining type 2 and type 3 pulses.

FIG. 6 shows mean quantitative expression data 48 hours after deliveringa plasmid coding for the luciferase reported molecule in vivo byapplication of pulsed fields.

FIGS. 7A-7F illustrate a proposed sequence of events occurring with thecombined use of types 1, 2, and 3 pulses.

FIG. 8 shows mean quantitative expression data for the luciferasereporter molecule in normal mouse muscle samples 2 days afterelectrically mediated delivery of plasmid coding for the luciferasecDNA.

FIGS. 9A-9G represent a proposed mechanism for the use of a type 2 pulseplus alternative administrations of the type 3 pulse with the same orwith a reversal of polarity.

FIG. 10 shows mean quantitative expression data for the luciferasereporter molecule in normal mouse muscle samples 7 days afterelectrically mediated delivery of plasmid coding for the luciferasecDNA, with the use of varied waveforms for the type 3 pulse.

FIG. 11 plots the waveforms of the pulses used for a delivery experimentin which the type 2 pulses were rectangular, but the fifth type 3 pulsecontained an exponentially rising component in the latter stages of thepulse.

FIG. 12 shows mean quantitative expression data for the luciferasereporter molecule in normal mouse muscle samples 48 h after electricallymediated delivery of plasmid coding for the luciferase cDNA, with theuse of sequential type 1 and type 2 pulses.

FIGS. 13A-13E represent a proposed mechanism for the use of a type 1pulse followed by a type 2 pulse.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A description of the preferred embodiments of the present invention willnow be presented with reference to FIGS. 3-13E.

A schematic diagram of an exemplary pulse sequence is illustrated inFIG. 3. An exemplary device for delivering pulses comprises a commercialdevice for producing dc pulses with characteristics needed as describedherein (CytoPulse PA-4000, CytoPulse Sciences, Hanover, MD). The termfield strength is used herein to indicate voltage, as is understood bythose of skill in the art. Field strength is the voltage applied to thecells/tissue divided by the distance between the electrodes. Theduration of each pulse, the time interval between pulses, and the numberof pulses are all variables in the pulse sequence of the presentinvention. While the waveforms shown in FIG. 3 are dipicted asrectangular, this pulse shape is not intended as a limitation, as otherwaveforms can also be used. For example, at least one of the first andthe second pulse may be selected from the group consisting of arectangular direct current pulse, an alternating current pulse, a pulsedalternating current pulse, an alternating current pulse having variablefrequency and amplitude, a variable direct current waveform, a variablealternating current pulse biased with constant direct current, a pulsehaving a triangular waveform, a pulse having exponentially risingcomponent, and a pulse having an exponentially decaying component.

A typical sequence for practicing the present invention comprises thesteps of:

1. Deliver desired molecules into the target tissue into an interstitialspace by a method such as is known in the art, including one or acombination of local injection, intratissue injection, systemicinjection, intravenous injection, intraarterial injection, particle 10bombardment, and jet injection.

2. Apply two or more suitable electrodes to a segment of or the entiretissue.

3. Apply at least one low-intensity pulse having an intensityinsufficient to cause membrane breakdown but sufficient to induce anelectrophoretic migration of molecules within the interstitial tissuespace (shown as two pulses labeled 31 in FIG. 3): This pulse can alsoforce charged molecules to adhere to cell membranes. Pulse duration maybe in the range of microseconds to seconds, or multiple seriallyadministered pulses can be provided, their sum comprising the desiredpulse duration. Field strength for this pulse is dependent upon thecell/tissue type but can range from 0 to 2000 V/cm.

4. After sufficient electrically facilitated movement within theinterstitial space and/or molecule adherence to the target cells, one ormore electric pulses having characteristics causing a temporarybreakdown of the cell membrane are applied to the tissue/cells via theelectrodes (shown as two pulses labeled 32 in FIG. 3). Although theexact characteristics of the pulses vary with cell/tissue type, pulseswith field strengths in the range of 50-10,000 V/cm with pulse durationsranging from 1 μs to seconds are required for cell permeabilization. Inrelative terms, pulses that induce electroporation have a higher fieldstrength than those used in (3) above. An administration of these pulsesforms pathways in the membrane through which extracellular molecules canenter the cytosol.

5. Delivery can be further enhanced by molecule movement after (4)because cells that have been treated as in (4) remain in a permeabilizedstate for times on the order of minutes to an hour. Molecular movementcan be induced electrophoretically by applying pulses that havecharacteristics similar to but not necessarily identical to those in(3), where, as was described, the pulse(s) can comprise a sum ofmultiple shorter pulses (shown as two pulses labeled 33 in FIG. 3). Thisincrease in the quantity of molecules internalized by the cellspresumably results from an electrophoretic movement of molecules fromthe extracellular space through the permeabilized cell membrane and intothe cell interior.

The above-listed steps comprise a typical scenario including pulseshaving different characteristics for three basic functions:

A first type of pulse is used as in (3) to cause electrophoreticmigration of a molecule within a tissue interstitial space and/ormolecule adherence to a target cell. A second type of pulse as in (4) tocause electropermeabilization of a target cell membrane. A third type ofpulse is used as in (5) to cause electrophoretic molecular movement fromthe extracellular space into the cell interior.

The system of the present invention may be used as follows:

1. Use electrophoresis pulses to cause molecular movement and/ormolecule adherence followed by electroporation pulses only (type 1 andtype 2 pulses).

2. Use electroporation pulses followed by electrophoresis pulses to movemolecules into electroporated cells only (type 2 and type 3 pulses).

3. Use electrophoresis pulses only to move and/or distribute moleculesthroughout the tissue (type 1 pulses).

4. Move/distribute/deliver more than one type of molecule simultaneously(any combination of types 1, 2, and 3 pulses).

5. Use electric pulsing protocols as described herein to move and/ordeliver two or more types of molecules that chemically react in vivo.Such a reaction can occur within the extracellular space or in theintracellular space (any combination of types 1, 2, and 3 pulses).

6. Use types of pulses other than rectangular direct current, such asalternating current, pulsed alternating current, high- and low-voltagealternating current with variable frequency and amplitude, variabledirect current waveforms, variable alternating current signals biasedwith variable direct current waveforms, variable alternating currentsignals biased with constant direct current. Use alternate waveformshapes such as triangular, sawtooth, exponentially rising, exponentiallydecaying, etc., as can be conceived by one of skill in the art.

7. Move, distribute, and deliver molecules using the pulsing scenariosdescribed herein such as amino acids, bioactive molecules, polypeptides,proteins, antibodies (or fractions thereof), glycoproteins, enzymes,nucleic acids, oligonucleotides, RNA, DNA, competent DNA, plasmid DNA,chromosomes, drugs, other organic and inorganic molecules that have alocalized charge region, other organic and inorganic molecules that donot contain a localized charged region, and any molecule modified tocontain a charged region.

Combined Use of Type 2 and Type 3 Pulses

Mean quantitative expression data for luciferase in normal murine skin 2days after delivery of a plasmid coding for luciferase cDNA using pulsedelectric fields are shown in FIG. 4. The data indicate skin samples thatwere treated in four different ways, having received an intradermalinjection of DNA.

1. DNA followed by no pulses.

2. DNA followed by pulses that were 1500 V/cm and 100 μs in duration(type 2 pulses).

3. DNA followed by pulses that were 100 V/cm and 20 ms in duration (type3 pulses).

4. DNA followed by 750 V/cm pulses that were 50 μs in duration and 40V/cm pulses that were 20 ms in duration (combined type 2 and type 3pulses).

Method (4) achieved very high expression, while very little expressionwas obtained in samples treated with 1500 or 100 V/cm pulses (or nopulses). This example indicates the efficacy of using two differenttypes of pulses, with higher expression than obtained for the sum ofseparately administered type 2 and type 3 pulses, even though the type 2pulse in (4) had a lower field strength and duration (750 V/cm, 50 μs)than the type 2 pulses used alone (1500 V/cm, 100 μs). Also, the type 3pulses used in the combined treatment (40 V/cm) provided lesselectrophoretic driving force than the type 3 pulses used alone (100V/cm).

A possible explanation for this synergistic effect may be as follows andas illustrated by FIGS. 5A-5E, although this is not intended as alimitation: A cell with a resting potential in a conductive medium ispositioned between two electrodes (FIG. 5A). A molecule is introducedinto the tissue (plasmid DNA, for example, is negatively charged insolution; FIG. SB).

Type 2 pulses are then applied to the cells to induce electroporation(FIG. SC). A rapid depolarization of the cell membrane results, causingan exchange of molecules on either side of the membrane.

Next type 3 pulses are applied to electrophoretically drive thenegatively charged plasmid DNA molecules into the electroporated cells,with the migration toward the positively charged electrode (FIG. 5D).Thus the addition of type 3 pulses may serve to enhance expression byelectrophoretically moving more plasmid DNA into cells during delivery.Minutes to hours after pulsation, the cell membrane reseals by normalmembrane fluidity, leaving the plasmid DNA inside the cell (FIG. 5E).

Combined Use of Type 2 and Type 3 Pulses and of Types 1, 2, and 3 Pulses

Mean quantitative expression data 48 hours after using electric fieldsto deliver a plasmid coding for the luciferase reporter molecule in rathepatocellular carcinomas in vivo are shown in FIG. 6. Traditionalsyringe and needle injection was used to administer a solution of theplasmid directly into a tumor prior to electrical treatment. Animals ineach of the groups (bars) represented in FIG. 6 were treated as follows:

1. DNA and no pulses.

2. DNA followed by 1500 V/cm pulses that were 100 μs in duration (type 2pulses; the currently used standard).

3. DNA followed by 500 V/cm pulses that were 100 μs in duration (type 2pulses).

4. DNA followed by 200 V/cm pulses that were 10 ms in duration (type 3pulses).

5. DNA followed by 500 V/cm pulses that were 100 μs in duration andpulses that were 200 V/cm with a duration of 10 ms (combined type 2 andtype 3 pulses).

6. DNA followed by 30 V/cm pulses that were 10 ms in duration, 750 V/cmpulses that were 100 μs in duration, and 30 V/cm pulses that were 10 msin duration (combined types 1, 2, and 3 pulses).

Luciferase expression for 1500 V/cm pulses of 100 μs duration (2) is thecurrently accepted standard for delivering molecules in vivo. Higherexpression can be obtained even when electroporation pulses with a lowerfield strength (500 V/cm) were used and combined with 200 V/cmelectrophoretic pulses (5). The magnitude of the expression was greaterthan the sum of the magnitudes of the two components when used alone(3+4). One potential mechanism for this synergy has already beendescribed.

The data for method (6) indicate a result similar to the currentstandard. A proposed mechanism for this is as follows and is illustratedin FIGS. 7A-7F, although this is not intended as a limitation: A cellwith a resting potential in a conductive medium is placed between twoelectrodes (FIG. 7A). A molecule is introduced into the tissue (FIG.7B). Pulses of type 1 are applied to electrophoretically distribute DNAthroughout the interstitial spaces (FIG. 7C). Since these pulses areinsufficient to cause cell membrane electroporation, the applied fieldinduces a polarized state in the cell, which could attract negativelycharged DNA molecules to the side of the cell facing the negativelycharged electrode. This may cause an electrostatic adhesion of the DNAmolecules to the cell membrane, which by proximity would provide agreater chance for DNA to enter the cell after electroporation has beenachieved in the next step.

Next type 2 pulses are applied to cause electroporation (FIG. 7D). Arapid depolarization of the cell membrane results, causing an exchangeof molecules on either side of the membrane. Pulses of type 3 are thenapplied (FIG. 7E), causing an electrophoretic movement of the negativelycharged molecules through the permeabihzed membrane surface facing thenegative electrode. Such a movement could potentially introduce moreplasmid DNA into each cell, thereby increasing expression.

Minutes to hours after pulsation, the cell membrane reseals by normalmembrane fluidity, leaving the plasmid DNA inside the cell (FIG. 7F).

Variation in the, Combined Use of Pulse Type 2 and Type 3

The preceding two examples illustrate a combined use of pulse types 2and 3. In these examples the positive and negative electrodes were inthe same physical orientation for both types of administered pulses. Analternate method of applying pulse types 2 and 3 involves switching theorientation of the positive and negative electrodes after type 2 pulseshave been administered but before type 3 pulses have been administered.

FIG. 8 shows mean quantitative expression data for the luciferasereporter molecule in normal mouse muscle samples 2 days afterelectrically mediated delivery of plasmid coding for the luciferasecDNA. A solution of plasmid DNA was introduced into the muscle tissue byinjection prior to electrically treating the muscle in four differentmanners:

1. DNA and no pulses.

2. DNA followed by 1500 V/cm pulses that were 100 μs in duration (type 2pulses).

3. DNA followed by 750 V/cm pulses that were 50 μs in duration and 40V/cm pulses that were 20 ms in duration (combined type 2 and type 3pulses). These pulses were administered with the same polarity.

4. DNA followed by 750 V/cm pulses that were 50 μs in duration and 40V/cm pulses that were 20 ms in duration (combined type 2 and type 3pulses). The type 2 and type 3 pulses were administered with oppositepolarity.

The current state of the art is represented by (2), and varying type 2and type 3 pulses by (3) as in previous examples. The data for (4),however, indicate that changing the polarity further enhances theexpression.

A possible explanation of the phenomenon of this example is as followsand as illustrated in FIGS. 9A-9G, although this is not intended as alimitation:

The cell is placed between two electrodes in a conductive medium (FIG.9A). A molecule is introduced into the tissue (FIG. 9B). Type 2 pulsesare applied to cause electroporation (FIG. 9C). A rapid depolarizationof the cell membrane results, causing an exchange of molecules on eitherside of the membrane.

Type 3 pulses are applied to cause an electrophoretic movement of thenegatively charged molecules through the permeabilized membrane surfacefacing the negative electrode as in (3) above (FIG. 9D). Alternatively,as in (4) above, the polarity of the type 3 pulses is opposite (FIG.9E). This causes electrophoretic movement of the negatively charged DNAmolecules through the opposite side of the cell. This opposite side ispreferentially more permeabilized when type 2 pulses are applied. Anadvantage to applying type 3 pulses in this manner is that the desiredmolecule, such as a DNA molecule, may be electrophoretically movedthrough an area of membrane that is more permeabilized, which wouldresult in more mass transport than if the type 2 and type 3 pulses wereapplied with the same polarity.

Minutes to hours after pulsation, the cell membrane reseals by normalmembrane fluidity, leaving the plasmid DNA inside the cell (FIG. 9F).Potentially, more DNA is transferred by switching polarity between types2 and 3 pulses to drive the molecules through the side of the cell thathas been porated to a greater extent (FIG. 9G).

Variation of the Waveform Used for Type 3 Pulses During the Combined Useof Types 2 and 3 Pulses

Mean quantitative expression data for the luciferase reporter moleculein normal mouse muscle samples 7 days after electrically mediateddelivery of plasmid coding for the luciferase cDNA are shown in FIG. 10.Samples were treated in eight different manners:

1. DNA and no pulses.

2. DNA followed by 1500 V/cm pulses that were 100 μs in duration (type 2pulses).

3. DNA followed by 750 V/cm pulses that were 50 μs in duration (type 2pulses).

4. DNA followed by 40 V/cm pulses that were 20 ms in duration (type 3pulses).

5. DNA followed by 14 V/cm pulses that were 20 ms in duration (type 3pulses).

6. DNA followed by 750 V/cm pulses that were 50 μs in duration andpulses that were 40 V/cm with a duration of 20 ms (combined types 2 and3 pulses).

7. DNA followed by 750 V/cm pulses that were 50 μs in duration andpulses that were 14 V/cm with a duration of 14 ms (combined types 2 and3 pulses).

Two type 2 pulses, in series, were delivered for each of the treatmentconditions that used the type of pulses above. Five type 3 pulses weredelivered in series for those treatment conditions that used type 3pulses. The difference between this example and the three previous oneswas in the waveform of the pulses. Whereas in the previous examplesrectangular pulses were used for both types 2 and 3 pulses, here thetype 2 pulses were rectangular, but the fifth type 3 pulse contained anexponentially rising component in the latter stages of the pulseindicated in FIG. 11.

The results of this example indicate that combining type 2 and type 3pulses causes an increase in luciferase expression in muscle samplesrelative to the sum of the expression from the samples that receivedeither type 2 or type 3 pulses alone. A possible explanation for thissynergistic effect is similar, to that given above, except that thewaveform for the postelectroporation electrophoresis is different,indicating that alternative waveforms to the rectangular shape can bebeneficial for this type of delivery.

Combined Use of Type 1 and Type 2 Pulses

Mean quantitative expression data for the luciferase reporter moleculein normal mouse muscle samples 48 h after electrically mediated deliveryof plasmid coding for the luciferase cDNA are shown in FIG. 12. Sampleswere treated in four different manners:

1. DNA and no pulses.

2. DNA followed by 2000 V/cm pulses that were 25 μs in duration (type 1pulses).

3. DNA followed by 60 V/cm pulses that were 10 ms in duration (type 2pulses).

4. DNA followed by 60 V/cm pulses that were 10 ms in duration (type 1pulses) and 2000 V/cm pulses that were 25 μs in duration (combined types1 and 2 pulses).

Luciferase expression for (4), which utilized combined types 1 and 2pulses, was higher than (2) and (3), which utilized a single type ofpulse. In addition, expression for (4) was higher than the sum of theexpression levels of (2) and (3).

These data indicate that using type 1 pulses with the traditional pulsesused for electroporation (type 2) can augment the resulting expression.It is believed that the combined use of types 1 and 2 pulses has not yetbeen optimized, and that additional synergy may result in anoptimization of the augmenting effect of the combined pulses.

A potential mechanism for this effect is depicted in FIGS. 13A-13E. InFIG. 13A, a cell with a resting potential is placed in a conductivemedium between two electrodes. A molecule is then introduced into thetissue (FIG. 13B), here plasmid DNA, which is negatively charged insolution.

Next pulses of type 1 are applied to electrophoretically distribute theDNA throughout the interstitial space (FIG. 13C). Since this pulse isnot sufficient to cause cell membrane electroporation, the applied fieldinduces a polarized state in the cell, attracting negatively chargedmolecules to the side of the cell facing the negatively chargedelectrode. Electrostatic adhesion of the molecules to the cell membraneis caused, enhancing the molecule's proximity to the membrane and henceits chance for entry following the subsequent electroporation step.

Pulses of type 2 are applied to cause electroporation (FIG. 13D). Arapid depolarization of the cell membrane results, causing an exchangeof molecules on either side of the membrane. Minutes to hours afterpulsation, the cell membrane reseals by normal membrane fluidity,leaving the molecule inside the cell (FIG. 1 3E).

It may be appreciated by one skilled in the art that additionalembodiments may be contemplated, including alternate waveforms andpulsing sequences.

In the foregoing description, certain terms have been used for brevity,clarity, and understanding, but no unnecessary limitations are to beimplied therefrom beyond the requirements of the prior art, because suchwords are used for description purposes herein and are intended to bebroadly construed. Moreover, the embodiments of the apparatusillustrated and described herein are by way of example, and the scope ofthe invention is not limited to the exact details of construction.

Having now described the invention, the construction, the operation anduse of preferred embodiment thereof, and the advantageous new and usefulresults obtained theeby the new and useful constructions, and reasonablemechanical equivalents thereof obvious to those skilled in the art, areset forth in the appended claims.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween. Now that theinvention has been described,

1. A method for delivering a charged molecule into a cell in vivocomprising the steps of: positioning a charged molecule outside andgenerally adjacent to the cell in vivo, wherein the cell comprises aconstituent of a tissue; delivering a first electromagnetic pulse to thecell having a strength and duration sufficient to cause electroporationof the cell; and delivering a second electromagnetic pulse to the cellhaving a strength and duration insufficient to cause electroporation ofthe cell and sufficient to cause an electromigration of the moleculetoward and into the cell, wherein at least one of the first pulse andthe second pulse comprises an exponentially rising component.
 2. Themethod recited in claim 1, wherein the molecule positioning stepcomprises the step of delivering the molecule into an interstitial spaceadjacent the cell.
 3. The method recited in claim 1, further comprisingthe step of placing a pair of electrodes with opposite polarity adjacentthe tissue and in spaced-apart relation from each other, and wherein thepulse delivering steps comprise administering the pulses via theelectrode pair.
 4. The method recited in claim 3, further comprising thestep of delivering a third electromagnetic pulse prior to the firstpulse, the third pulse having a strength and duration insufficient tocause electroporation of the cell and sufficient to cause anelectromigration of the molecule toward the cell, wherein at least oneof the first pulse, the second pulse and the third pulse comprises anexponentially rising field strength component.
 5. The method recited inclaim 3, wherein during delivery of the first pulse a first of theelectrode pair has a first polarity and a second of the electrode pairhas a second polarity opposite the first polarity and during delivery ofthe second pulse the first electrode has the second polarity and thesecond electrode has the first polarity.
 6. The method recited in claim1, wherein the second pulse has a field strength in a range of 1 to 2000V/cm.
 7. The method recited in claim 1, wherein the first pulse has afield strength in a range of 50 to 10,000 V/cm.
 8. The method recited inclaim 7, wherein the first pulse has a duration of at least 1 μs.
 9. Themethod recited in claim 1, wherein at least one of the first and thesecond pulse comprises a series of pulses.
 10. The method recited inclaim 1, wherein the first pulse is delivered with a first polarity andthe second pulse is delivered with a second polarity opposite the firstpolarity.
 11. The method recited in claim 1, wherein the chargedmolecule positioning step comprises a step selected from the groupconsisting of injection, particle bombardment, and jet injection. 12.The method recited in claim 1, wherein the charged molecule comprises aplurality of different charged molecules.